We have associated over-expression of the glucose-regulated stress protein, GRP78, with increased sensitivity to DNA cross-linking agents and with the impairment of DNA cross-link repair. This sensitization, first shown in V79 hamster cells, was extended to three human tumor cell lines each bearing a different genetic alteration. Under these conditions, tumor cells deficient in p53, alkylguanine transferase or the mismatch repair protein, hMLH1, were sensitized to BCNU, cisplatin or melphalan. This is important, first, since it is known that in many cases, tumors bearing genetic mutations are resistant to chemotherapy, a means of sensitizing these resistant cells would be of great interest. Second, in direct contrast to the conventional approach of targeting therapy to a single genetic deficiency this approach may lead to an alternative direction in therapy. The thrust of this application is to determine the mechanisms underlying the association of GRP78 up-regulation with hypersensitivity to cross-linking agents. We propose to examine the following plausible mechanism of sensitization: It has been reported that when GRP78 is over-expressed, it moves from the endoplasmic reticulum to the cell nucleus. In character with its nature as a binding protein, GRP78 binds to one or more DNA repair enzymes involved in cross-link repair and thus explain the increased sensitivity to cross-linking agents. We propose to confirm the nuclear localization of GRP78 in human cancer cell lines by immunofluorescence techniques and to link the nuclear localization of GRP78 in human cancer cell lines by immunofluorescence techniques and to link the nuclear localization of GRPF78 to increased sensitivity to cross-linking agents as well as to reduced levels and activity of DNA repair enzymes. We will use several complementary systems to induce or suppress the expression of GRP78 in order to link the protein directly with alterations of elements of DNA repair. We will measure the cellular responses to cross-linking agents under stress conditions via several indices of nucleotide excision repair efficacy. Finally, we will examine whether GRP78 binds directly to selected DNA repair enzymes by co-immunoprecipitation. A basic understanding of the mechanisms behind the sensitization of cells during GRP78 up-regulation may provide a new direction in improving the efficacy of cancer chemotherapy.